EVPS is focused on the development and characterization of authentic animal models of disease for orthopoxviruses and hemorrhagic fever viruses and the identification of countermeasures against disease caused by these infections. Findings from FY09 are summarized below. 1. Viral pathogenesis of orthopoxviruses and development of countermeasures against infection. Identification of an animal model that most closely resembles natural smallpox transmission and infection is critically important for testing of countermeasures because efficacy studies in humans are obviously not possible. We have investigated both monkeypox virus (MPXV) and cowpox virus (CPXV) infection of nonhuman primates (NHPs). Comparison of the intravenous (i.v.) and intrabronchial (i.b.) route of MPXV infection was studied to determine if i.b. inoculation would result in disease that more closely resembles the course of naturally acquired orthopoxvirus infection via the aerosol route. Uniform lethal disease was observed with an i.v. challenge dose of 5x10e7 PFU in cynomolgus macaques, while a ten-fold reduced challenge resulted in 33% moribundity. Clinical signs consisted of extensive lesion development, fever, and anorexia. A pro-inflammatory cytokine response was observed in NHPs as demonstrated by increase levels of IL-6, IL-8, and MCP-1. In contrast, i.b. infection only resulted in 33% moribundity at the highest dose tested although lesional disease and fever were observed. Viral load was similar between the i.v. and i.b. groups. Although disease was observed, we found significant consolidation of the lung and severe broncho-pneumonia at the site of inoculation which may limit the usefulness of this model. We have also employed CT and PET/CT imaging to monitor progression of disease after i.b. inoculation and observed lymph node inflammation and consolidation and inflammation of the lung. We are currently planning to use the i.v. route for study of countermeasures and considering further routes of inoculation including aerosol. We have further pursued CPXV infection as a BSL-2 model of orthopoxvirus infection and a surrogate model of variola virus infection and demonstrated that i.b. infection with CPXV Brighton strain replicates to high levels in NHPs resulting in lethal disease. A new CPXV strain derived from a lethal outbreak in a German primate center in 2002 was also evaluated in NHPs because it was suspected to be highly virulent. Surprisingly, only mild disease with low fever and no moribundity was observed. Nevertheless, CPXV Brighton infection of NHPs has been established as a potential model of orthopoxvirus infection. Our initial investigation of antipoxviral drugs has focused on Mitoxantrone which is an approved chemotherapy drug that was demonstrated to have in vitro antiviral activity against vaccinia virus. We found that Mitoxantrone had an EC50 of 0.03 uM against CPXV and 0.4 uM against MPXV which were comparable to the results observed previously with vaccinia virus. In a virus yield assay using the CPXV Brighton strain, the presence of mitoxantrone was able to block virus replication as determined by plaque assay of tissue culture supernatants. In contrast, MPXV was only marginally inhibited by mitoxantrone in a virus yield assay. We found that treatment of CPXV-infected mice with 0.5 mg/kg mitoxantrone resulted in 25% protection from moribundity and an extension of 3.5 days in mean time of death of nonsurvivors compared to untreated controls. We have demonstrated limited efficacy of mitoxantrone in vivo against CPXV indicating that this drug might be pursued further alone or in combination with other poxviral treatments. 2. Establishment of simian hemorrhagic fever virus as a BSL-2 model of viral hemorrhagic fever. Simian hemorrhagic fever virus (SHFV), a member of the arterivirus family, is classified as a BSL-2 agent as it is not infectious to humans and can therefore be used to study the pathogenesis of viral hemorrhagic fevers in a NHP model without BSL-4 containment. The virus was shown to be responsible for explosive outbreaks of disease in NHP facilities in the 1960s to 1980s before it was determined that epizootics could be blocked by preventative measures including improved aseptic technique and sequestration of species. We inoculated groups of three rhesus macaques with serial ten-fold dilutions of SHFV from 5x10e4 PFU to 5x10e1 PFU and observed that severity of disease was not dose-dependent. NHPs exhibited clinical signs similar to previous reports of SHFV infection including fever, anorexia, increased clotting time, mild facial edema, mild petechial rash, and increased ALP, AST, and ALT values. Approximately 66% of NHPs across the dosage groups succumbed to infection with average time to moribundity of 10 to 16 days. Coagulation defects were detected including increased APTT clotting times. Increased levels of proinflammatory cytokines were detected after infection including interferon gamma, IL-8 and IL-12. Unexpectedly, nearly 50% of SHFV-infected NHPs had evidence of sepsis and secondary bacterial infection which is not typically associated with viral hemorrhagic fevers although it has been reported. We are currently testing whether a higher dose will result in more consistent disease and the reproducibility of the finding of sepsis. 3. Countermeasures against viral hemorrhagic fevers. A safe and effective vaccine against Zaire ebola virus (ZEBOV) would potentially be used by multiple groups including healthcare providers, laboratory researchers, and military personnel. A vaccine could also be used to mitigate outbreaks in Africa and possibly be used to protect endemic nonhuman primate populations that have been affected by ZEBOV. While multiple strategies for ZEBOV vaccine development have been pursued, no current vaccine is close to being licensed by the Food and Drug Administration. Unresolved issues with current vaccine candidates include safety concerns, pre-existing immunity to vectored vaccines, and manufacturing and dosage obstacles. We are developing an alternative vaccine approach in collaboration with Dr. Matthias Schnell using attenuated rabies virus vaccine based vectors that express the major protective antigen of ZEBOV, the virion surface glycoprotein (GP). Rabies virus based vectors have previously been generated that express protective antigens from human immunodeficiency virus, hepatitis C virus, and anthrax protective antigen and found to be safe, attenuated, and efficacious in mice and/or nonhuman primates. The main objective of the present study is to determine the level of immunogenicity of rabies virus vaccine-based vectors expressing ZEBOV GP as either live or killed vaccines and determine their efficacy in animal models of ZEBOV infection. Currently, we have generated an infectious cDNA clone of rabies vaccine virus expressing ZEBOV GP and recovered it in tissue culture. We will next expand the virus, characterize it in vitro, and investigate immunogenicity and efficacy in mice.